Figure 4.

Potent intermolecular complementation-dependent tumoricidal activity of the engineered PrAg proteins. B16-BL6 melanoma-bearing (a), T241 fibrosarcoma-bearing (d), and LL3 Lewis lung carcinoma-bearing (e) mice were injected intradermally adjacent to the tumor nodules with PBS (■), 6 μg PrAg-U2-R200A (▲), 6 μg PrAg-L1-I210A alone (◆), or a combination of 3 μg PrAg-U2-R200A and 3 μg PrAg-L1-I210A (●) in the presence of 0.5 μg FP59 at day 0, 3, and 6. (b-c) Microscopic appearance of B16-BL6 melanoma cells 24 h after a single injection of the mice with PBS or the mixture of 3 μg PrAg-U2-R200A, 3 μg PrAg-L1-I210A, and 0.5 μg FP59. Extensive tumor necrosis (empty arrow in c, H&E staining, 64×, and insert 160×) and regressive changes, such as karyopyknosis and karyorhexis (arrows in insert) are seen in toxin-treated but not mock-treated tumor (b, H&E staining, 64×, insert 160×). (f) Comparison of the anti-tumor efficacy of the mutated PrAg proteins. Five groups of B16-BL6 melanoma-bearing mice were treated intradermally with PBS, 2 μg or 6 μg of PrAg-U2 or the mixture of PrAg-U2-R200A/PrAg-L1-I210A (1 + 1 μg or 3 + 3 μg, respectively) in the presence of 0.5 μg FP59 at day 0, 3, and 6. Two groups of the tumor-bearing mice were treated intraperitoneally with PBS or the mixture of 20 μg PrAg-U2-R200A and 10 μg PrAg-L1-I210A in the presence of 3 μg FP59 at day 0, 3, and 6 (2/3 MTD3). The weight of intradermal tumor nodules is expressed as mean tumor weight ± the SEM. * in (a), (d), and (e) represents significance (p < 0.05) between the treatments using the combination of PrAg-U2-R200A and PrAg-L1-I210A and using PrAg-U2-R200A or PrAg-L1-I210A alone. * in (f) represents significance (p < 0.01) between the treatments via i.p. with PBS and the combination of PrAg-U2-R200A and PrAg-L1-I210A.